Biochemical characterisation of navicular hyaline cartilage, navicular fibrocartilage and the deep digital flexor tendon in horses with navicular disease

Research in Veterinary Science 75 (2003) 113–120 www.elsevier.com/locate/rvsc

Biochemical characterisation of navicular hyaline cartilage, navicular fibrocartilage and the deep digital flexor tendon in horses with navicular disease M. Viitanen *, J. Bird, R. Smith, R.-M. Tulamo, S.A. May Royal Veterinary College, FAEMS, University of London, London, UK Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, University of Helsinki, Helsinki, Finland Accepted 6 March 2003

Abstract The study hypothesis was that navicular disease is a process analogous to degenerative joint disease, which leads to changes in navicular fibrocartilage and in deep digital flexor tendon (DDFT) matrix composition and that the process extends to the adjacent distal interphalangeal joint. The objectives were to compare the biochemical composition of the navicular articular and palmar cartilages from 18 horses with navicular disease with 49 horses with no history of front limb lameness, and to compare navicular fibrocartilage with medial meniscus of the stifle and collateral cartilage of the hoof. Cartilage oligomeric matrix protein (COMP), deoxyribonucleic acid (DNA), total glycosaminoglycan (GAG), metalloproteinases MMP-2 and MMP-9 and water content in tissues were measured. Hyaline cartilage had the highest content of COMP and COMP content in hyaline cartilage and tendon was higher in lame horses than in sound horses (p < 0:05). The concentration of MMP-2 amount in hyaline cartilage was higher in lame horses than in sound horses. The MMP-2 amounts were significantly higher in tendons compared to other tissue types. Overall, 79% of the lame horses with lesions had MMP-9 in their tendons and the amount was higher than in sound horses (p < 0:05). In horses with navicular disease there were matrix changes in navicular hyaline and fibrocartilage as well as the DDFT with potential implications for the pathogenesis and management of the condition. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Navicular disease; Navicular hyaline cartilage; Navicular fibrocartilage

1. Introduction Navicular disease is estimated to account for onethird of all chronic forelimb lamenesses in horses (Colles, 1982). Despite the high incidence of this disease, and continuing research into the nature of the problem, the exact cause remains unknown. Currently there are two basic theories regarding the aetiology: the vascular theory and mechanical theory. Adams (1969) described navicular disease as a condition, which begins with bursitis of the navicular bursa, between the flexor tendon and the navicular bone, and ultimately leads to degenerative and erosive lesions of fibrocartilage. *

Corresponding author. Tel.: +358-505664862; fax: +358-98136994. E-mail address: [email protected] (M. Viitanen).

It is generally accepted that navicular disease is a degenerative disease involving the distal sesamoid (navicular bone), navicular bursa and deep digital flexor tendon (DDFT). In advanced cases of the disease, pathological findings include erosion and ulceration of the fibrocartilage on the flexor surface of the bone, and tearing of the DDFT in contact with the bone (Asquith and Kivipelto, 1994). No lesions of the navicular hyaline cartilage have been reported. Diagnosis is based on clinical examination, nerve blocks and radiographs. Radiographic changes, previously regarded as diagnostic of navicular disease (MacGregor, 1984), can also occur in horses without lameness (Ackerman et al., 1977), and radiographic signs correlate poorly with lameness (Wright, 1993). The diagnosis ‘‘navicular disease’’ is not always restricted to problems of the navicular area alone. The

0034-5288/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0034-5288(03)00072-9

114

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

disease may be diagnosed when the horse has a front limb lameness that is alleviated with a palmar digital nerve block (Stashak, 1987; Turner, 1986). However, a palmar digital nerve block alleviates pain arising from all structures in the palmar half of the foot, not only just the navicular bone (Dyson, 1986), so this leads to confusion particularly in comparing accounts relating to the success rates of different treatments. Intra-articular analgesia of the distal interphalangeal joint (DIP-joint) or the navicular bursa is often used to add further information on the origin of the pain. These techniques are not specific to the injected space only, and can block out pain from the sole of the foot (Dyson and Kidd, 1993; Keegan et al., 1996; Schumacher et al., 2000; Schumacher et al., 2001). Synovial fluid studies have shown that the results obtained from the navicular bursa and the DIP-joint are very similar in horses that are not lame (Viitanen et al., 2000). In horses with navicular disease, the relative activity of metalloproteinases was increased, and glycosaminoglycan (GAG) levels were decreased both in the synovial fluid of the DIP-joint and in bursal fluids (Viitanen et al., 2001). Understanding the molecular composition of the extracellular matrix will enable more light to be thrown on the aetiopathogenesis of navicular disease. Cartilage oligomeric matrix protein (COMP) is synthesised in ligament, tendon, meniscus, and articular cartilage (Murray et al., 2001). Studies in human have demonstrated that it may be used as a prognostic marker in rheumatoid arthritis and osteoarthritis. Mechanical loading (Murray et al., 2001) may influence the distribution of COMP in articular cartilage. COMP degradation in synovial fluids from advanced joint disease may be due to metalloproteinases (MMP), gelatinolytic activity (Misumi et al., 2001). Areas of involvement of MMPs in pathological processes include tissue destruction, fibrotic diseases and weakening of the matrix (Woessner, 1998). Cartilage destruction in osteoarthritis (OA) is associated with increased levels of several matrix MMPs, including the gelatinase MMP-2 (Clegg et al., 1997; Thompson et al., 2001). Activation of MMPs occurs in joint disease, and in vitro stimulation of equine articular cells and tissues causes not only an increase in MMP production, but also an increase in amount of activated enzyme released (Clegg and Carter, 1999). Studies of synovial fluid, from horses with navicular disease, collected from the navicular bursal fluid and DIP-joint, showed significant changes in bursal metalloproteinase activities, GAG and hyaluronan content (Viitanen et al., 2001). Different types of tissues are present in the navicular area and therefore changes in levels of MMPs or GAGs may not only be derived from cartilage, but also from tendon and ligaments. The hypothesis of this study accepts the greater complexity of tissue types present in the navicular area in

comparison to the synovial joint, proposes that navicular disease is a process analogous to degenerative joint disease (osteoarthritis) which leads to similar changes in navicular fibrocartilage and in DDFT matrix composition. The subsidiary hypothesis was that this process extends, simultaneously, to the adjacent synovial cavity, the DIP-joint, and navicular hyaline cartilage.

2. Materials and methods 2.1. Tissue source A total of 67 horses were used in this study. Samples were collected, after euthanasia, from 18 horses diagnosed clinically with navicular disease [having a mean age of 10.8 (0.5)]. Control horses without a history of front limb lameness comprised 34 middle-aged horses [5–14 years, mean 8.8 (0.6)], 8 old horses [>18 years, mean 22 (0.8)] and 7 young horses [<2 years, mean 0.9 (0.3)]. No differences between the sexes were observed in these experiments; therefore the data were not segregated on the basis of gender. Horses with navicular disease came from the Royal Veterinary College and an abattoir near Bristol. A history was obtained either at the time of the clinical examination carried out at the Royal Veterinary College or via a questionnaire sent to the owner of the horse. Horses were diagnosed as having navicular disease if they had been lame and had shown a positive response to a palmar digital nerve block. All diseased horses used in this study had a history of a chronic navicular problem (more than 2 years). Both front legs were examined in all but three diseased horses. The cartilages and tendon of the control groups used in this study were obtained from horses destroyed for reasons other than front limb lameness. Only one leg from each horse was used. Full thickness cartilage samples were harvested from the whole articular surface of the navicular hyaline cartilage and navicular fibrocartilage. DDFT samples were collected from the area opposite to the navicular bone (Fig. 1). Collateral cartilage was obtained from the medial side of the hoof, and the proximal part of the cartilage was collected. The medial meniscus of the stifle was also used for this study. Collateral cartilage and meniscus samples and samples analysed from old and young horses were collected from sound animals. Collateral cartilage and meniscus samples only came from middle-aged horses (5–15 years). Samples were assessed macroscopically, and stored at )20°C until analysed. Tissue wet-weights were taken before freezing. Navicular hyaline cartilage, navicular fibrocartilage and tendon samples from middle-aged horses were divided into four groups: 1, sound horses with no macroscopic lesions in the navicular fibrocartilage or tendon; 2, sound horses

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

115

Fig. 1. Diagrammatic representation of a sagittal section of the foot.

with macroscopic lesions in the navicular area; 3, lame horses with no macroscopic lesions in the navicular area; and 4, lame horses with macroscopic lesions in the navicular area. Lesions were assessed by eye, fraying of the cartilage and tendon surfaces were counted as a lesion. Slight yellowish discoloration in the navicular fibrocartilage or DDFT was considered to be a normal age-related sign, and was not counted as a lesion (Colles, 1979). All the horses in groups with lesions had changes in both the navicular fibrocartilage and DDFT. Throughout the text, these groups are referred to as lame and sound groups with or without lesions. 2.2. Determination of DNA content The cellularity of the tissues was determined by assessing the total DNA content of the tissue following papain digestion. DNA was quantified using the bisbenzimidazole fluorescent dye technique, Hoechst 33258 (Kim et al., 1988). The Hoechst 33258 dye was used at a concentration of 0.2 mg/ml, in 0.01 mol/L Tris, 1.0 mmol/L EDTA, and 0.1 mol/L NaCl. Fluorescence of aliquots of the digests was evaluated by spectrofluorimetry: emission in the range 400–550 nm for an excitation wavelength of 365 nm was determined. Standard curves were determined using solutions of highly polymerised calf thymus DNA of known concentration.

COMP in the supernatant was determined using a heterologous inhibition ELISA (Smith et al., 1997). 2.5. Determination of GAG Cartilage and tendon samples were digested with papain (25 mg/ml; Sigma type III), in 50 mmol/L sodium phosphate buffer, pH 6.5, 2.0 mmol/L EDTA, 2.0 mmol/ L N-acetylcysteine for a minimum of 6 h at 60 °C. The concentration of total glycosaminoglycan in the digests was determined by the dimethylene blue dye assay (Farndale et al., 1986). Standard curves for the assay were constructed using purified chondroitin sulphate from shark cartilage. Separation of protein fragments was achieved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Aliquots of GnHCl, ethanol precipitated extracts were analysed on a tissue wet weightequivalent basis, by Western blotting onto polyvinylidene fluoridene membrane. Detection of ÔaggrecanaseÕ and the metalloproteinase generated G1 domain of aggrecan was achieved using polyclonal antisera that specifically recognise the neo-carboxy terminal regions of the interglobular domain produced by their proteolytic activity (Lark et al., 1997). The antisera, which recognise the aggrecanase and metalloproteinase cleavage sites, were raised against NITEGE and FVDIPEN sequences, respectively (Lark et al., 1997).

2.3. Extraction of proteoglycan and protein 2.6. Determination of matrix metalloproteinases Cartilage and tendon was extracted twice with 4.0 mol/L guanidine hydrochloride (GnHCl) in 0.05 mol/L sodium acetate buffer, pH 5.8, containing proteinase inhibitors [pepstatin (1.0 lg/ml, 1,10-phenanthroline (1.0 mmol/L), iodoacetic acid (1.0 mmol/L), phenylmethylsulphonyl fluoride (1.0 mmol/L)] at 4 °C for 24 h (Platt and Bayliss, 1994). 2.4. Determination of COMP GnHCl extracts were precipitated twice with 95% ethanol, 50 mM sodium acetate ()20 °C overnight), centrifuged at 8000g for 30 min and freeze-dried. Pellets were resuspended with sample buffer and the quantity of

Relative amounts of MMP-2 and -9 were assayed by gelatin zymography (Sepper et al., 1994). Gelatin was polymerised in 8% polyacrylamide gels. Dry samples (3 mg) or wet samples (10 mg) of tissue were mixed with 100 ll sample buffer containing 0.471 M Tris, 0.256 mol/ L H3 PO4 , 20% glycerol, 0.04% bromophenol blue and 6% sodium dodecyl sulphate. Samples were heated for 30 min at 60 °C and 10 ll aliquots of each sample was loaded onto the gels. Polymorphonuclear neutrophils, separated from equine blood, were used as an internal standard in each gel (Raulo and Maisi, 1998). The intensity of the zymogen bands was compared to internal standard and assessed using computer assisted image

116

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

analysis of the gels (Raulo and Maisi, 1998). For each sample, the intensity of the enzyme band was calculated by comparison to the amount in the standard (neutrophils), thereby giving relative metalloproteinase amount. The standard was applied in each gel, enabling samples to be compared between gels (Clegg et al., 1997). 2.7. Determination of water content Portions of tissue of known wet weight were freeze dried for 24 h. The remaining tissue was measured and the water content calculated. 2.8. Statistical analysis Comparison of means between different groups was calculated using the one-way ANOVA test.

3. Results All results are presented in Tables 1–3. 3.1. Disease-related changes in navicular tissues Typically tissues with lesions contained less DNA than similar tissues without lesions but these differences were not statistically significant. No lesions in the navicular hyaline cartilage were noted in this study. However, hyaline cartilage from lame horses with lesions in the fibrocartilage had the

highest content of COMP, and this difference was statistically significant when compared to the hyaline cartilage from sound horses (p < 0:05). Tendons from lame horses with lesions had also significantly higher COMP values than tendons from sound horses (p < 0:05). Total GAG was calculated as a proportion of the wet weight of tissue. The highest GAG values were identified in hyaline cartilage from sound horses without lesions (p < 0:05). Hyaline cartilage from both sound and lame horses with lesions in the navicular fibrocartilage had significantly lower GAG values than the hyaline cartilage from horses without lesions (p < 0:05). Similarly, fibrocartilage of sound horses with no lesions had significantly higher GAG values than fibrocartilage of both sound and lame horses with lesions (p < 0:05). The presence of ÔaggrecanaseÕ and the metalloproteinase generated cleavage sites adjacent to the G1 domain of aggrecan were identified in all tissue types but no major differences in content were found between any of the groups. Navicular hyaline cartilage from lame horses with lesions had significantly higher MMP-2 values than that of sound horses (p < 0:05). Fibrocartilage of lame horses with lesions had higher MMP-2 amounts than that of sound horses but this difference was not statistically significant. Tendons of both lame and sound horses with lesions had significantly higher relative MMP-2 amounts when compared with the tendons of animals without lesions of the fibrocartilage (p < 0:05). Overall, 79% of the tendons of lame horses with lesions had MMP-9 present. In contrast, approximately

Table 1 Results of assays, mean (SE) of cartilage oligomeric matrix protein (COMP), glycosaminoglycan (GAG), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and water content in navicular hyaline cartilage Control, no lesions, N ¼ 27 DNA lg/mg of wet tissue COMP lg/mg of wet tissue GAG lg/mg of wet tissue MMP-2e relative amount MMP-9e relative amount Water%

Control, with lesions, N ¼ 7

Diseased, no lesions, N ¼ 8ð15Þd

Diseased, with lesions, N ¼ 10ð18Þd

Young horses, N ¼7

Old horses, N ¼8

0.23 (0.03)

0.11 (0.01)

0.26 (0.03)

0.18 (0.02)

0.2 (0.06)

0.26 (0.09)

2.7 (0.3)b

2.1 (0.7)

3.5 (1.3)

4.6 (1.0)a

0.9 (1.2)a

4.2 (1.2)a

52.2 (3.3)c

39.1 (2.9)a

44.6 (5.0)

40.5 (2.7)a

50.7 (3.9)

36.6 (3.1)a

3.1 (0.9)

1.8 (0.4)

3.6 (0.9)

9.3 (2.2)a

4.0 (2.1)

2.0 (0.9)

3.1 (1.2)

2.3 (0.6)

1.0 (0.4)

6.9 (2.1)

72.5 (0.5)

70.9 (0.6)

71.9 (0.8)

70.3 (1.7)

– 68.3 (3.4)

1.4 (0.5) 72.8 (0.4)

Hyaline cartilage of horses with navicular disease and macroscopic lesions in navicular fibrocartilage, had significantly more COMP and MMP-2 and less GAG than hyaline cartilage of sound horses. In navicular hyaline cartilage, COMP content increased and GAG content decreased with age. a p < 0:05, in comparison with sound horses without fibrocartilage lesions. b COMP values were significantly higher in hyaline cartilage than in any other tissues analysed (p < 0:05). c GAG values were significantly higher in hyaline cartilage of sound horses than rest of the tissues except hyaline and fibrocartilage of young horses (p < 0:05). d N is number of horses and number in brackets describes individual samples from which means were derived. e The relative amount of metalloproteinases was compared between samples. To determine the amounts of metalloproteinase, a neutrophil standard was applied to each gel, and the samples compared against it, and each other.

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

117

Table 2 Results of assays, mean (SE) of cartilage oligomeric matrix protein (COMP), glycosaminoglycan (GAG), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and water content in navicular fibrocartilage, meniscus and collateral cartilage Fibrocartilage

DNA lg/mg of wet tissue COMP lg/mg of wet tissue GAG lg/mg of wet tissue MMP-2 relative amountf MMP-9 relative amountf Water%

Diseased, no lesions, N ¼ 8ð15Þe

Meniscus N ¼ 10

Collateral cartilage N ¼ 12

Control, no lesions, N ¼ 27

Control, with lesions, N ¼7

0.3 (0.05)

0.29 (0.06)

0.29 (0.04)

0.25 (0.01)

0.47 (0.2)

0.33 (0.04)

0.46 (0.1)b

0.21 (0.02)

1.5 (0.2)

0.8 (0.3)

1.4 (0.3)

2.0 (0.7)

0.6 (0.3)

2.0 (0.4)

2.5 (0.8)

1.5 (0.4)

43.5 (3.1)

25.2 (3.4)a

38.1 (3.7)

33.8 (2.1)a

46.6 (6.4)c

35.8 (6.7)a

34.8 (5.5)

42.2 (4.0)

5.1 (0.9)

3.2 (0.2)

3.7 (0.6)

8.5 (2.5)

5.6 (1.1)

4.2 (0.8)

1.8 (0.2)

1.5 (0.3)

2.8 (1.1)

4.0 (1.7)

1.4 (0.5)

3.1 (1.2)

71.2 (1.2)

70.5 (0.9)

72.1 (0.8)

67.1 (1.9)

Diseased, with lesions, N ¼ 10ð18Þe

Young horses, N ¼7

– 69.7 (3.5)

Old horses, N ¼8

3.1 (1.9) 70.8 (1.9)

–

–

61.3 (1.2)d

67 (1.9)

Middle aged horses (5–15 years) were the source of meniscus and collateral cartilage. GAG content decreased both in sound and navicular diseased horses with fibrocartilage lesions. GAG content also decreased with age. DNA content was highest and the water content lowest in meniscus. a p < 0:05, in comparison with sound horses without fibrocartilage lesions. b The DNA content was higher in meniscus than in navicular hyaline or fibrocartilage or in collateral cartilage (p < 0:05). c Navicular fibrocartilage of young horses had higher GAG content than the navicular fibrocartilage of old horses (p < 0:05). d Water content in meniscus was lower than in navicular hyaline or fibrocartilage or in collateral cartilage p < 0:05. e N is number of horses and number in brackets describes individual samples from which means were derived. f The relative amount of metalloproteinases was compared between samples. To determine the amounts of metalloproteinase, a neutrophil standard was applied to each gel, and the samples compared against it, and each other.

Table 3 Results of assays, mean (SE) of cartilage oligomeric matrix protein (COMP), glycosaminoglycan (GAG), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and water content in deep digital flexor tendon (DDFT) Control, no lesions, N ¼ 27 DNA lg/mg of wet tissue COMP lg/mg of wet tissue GAG lg/mg of wet tissue MMP-2 relative amountg MMP-9 relative amountg Water%

Control, with lesions, N ¼ 7

Diseased, no lesions, N ¼ 8ð15Þf

Diseased, with lesions, N ¼ 10ð18Þf

Young horses, N ¼7

Old horses, N ¼8

0.33 (0.05)b

0.33 (0.08)

0.35 (0.03)

0.33 (0.04)

0.46 (0.1)

0.41 (0.06)

1.7 (0.4)

0.6 (0.1)a

2.0 (0.5)

3.1 (0.7)

0.3 (0.05)a

3.2 (0.7)

26.3 (2)c

26.4 (6.3)

24.9 (2.8)

28.0 (5.0)

21.7 (5.2)d

37.5 (7.5)a

16.9 (3)a

34.7 (5.1)a

4.1 (1.1)

9.4 (2.7)

15.9 (5.3)a

25.6 (12.5)a

–

61.1 (1.6)e

60.9 (0.9)

65.2 (1.0)

63.9 (0.9)

61.1 (0.9)

30.7 (3.4) 4.3 (1.7)a

26.6 (4) 3.8 (1.4)a 6.9 (2.1) 63.6 (1.6)

Relative amount of MMP-9 was increased in horses with navicular disease. Both sound horses and navicular diseased horses with lesions had higher MMP-2 amount than horses without lesions. Relative amount of MMP-2 was highest in the DDFT than other tissue types analysed. a p < 0:05, in comparison with sound horses without lesions. b The DNA content per unit weight of tissue was higher in the DDFT when compared to navicular hyaline cartilage and collateral cartilage p < 0:05. c DDFT had less GAG than collateral cartilage and fibrocartilage (p < 0:05). d DDFT had higher MMP-2 amounts than other tissue types analysed p < 0:05. e Water content was lower in DDFT than in navicular hyaline or fibrocartilage or collateral cartilage. f N is number of horses and number in brackets describes individual samples from which means were derived. g The relative amount of metalloproteinases was compared between samples. To determine the amounts of metalloproteinase, a neutrophil standard was applied to each gel, and the samples compared against it, and each other.

40% of all tissues from sound horses had MMP-9 present. Approximately 50% of hyaline and fibrocartilage from lame horses had MMP-9 present. The MMP-9

amounts in tendons of lame horses with lesions were significantly higher than in tendons of sound horses without lesions (p < 0:05).

118

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

The water content in navicular hyaline and fibrocartilage was approximately 70% and did not significantly vary between sound and lame horses. 3.2. Age-related biochemical changes The navicular fibrocartilage of young horses had a higher cellularity when compared with any hyaline cartilage (p < 0:05). The GAG content decreased with age in both navicular hyaline and fibrocartilage (p < 0:05). Interestingly, the only statistically significant age-related change in MMP-2 was a peak in tendons from middle-aged horses (p < 0:05). No measurable MMP-9 was present in young horses, whereas approximately 50% of old animals had MMP-9 in the DDFT. 3.3. Comparison of fibrocartilages The DNA content per unit weight of cartilage, a measure of the tissue cellularity, was higher in meniscus and tendon when compared with navicular hyaline cartilage and collateral cartilage (p < 0:05). Navicular fibrocartilages had more DNA than hyaline cartilages but these differences were not significant. When comparing different types of fibrocartilage, the meniscus had a higher COMP content than collateral cartilage and navicular fibrocartilage but this difference was not statistically significant. COMP values were significantly higher in navicular hyaline cartilage than in other tissues (p < 0:05). GAG content of the fibrocartilages was the highest in collateral cartilage and lowest in tendon. Navicular fibrocartilage and the meniscus had similar GAG contents. Differences were statistically significant when comparing collateral cartilage and fibrocartilage to the tendons (p < 0:05). When comparing tendons, fibrocartilage, collateral cartilage and meniscus, it was noted that the relative MMP-2 amount was highest in tendons and that navicular fibrocartilage had higher activity levels than meniscus and collateral cartilage. These differences were statistically significant only for tendons (p < 0:05). There was no MMP-9 detected in adult collateral cartilage or the meniscus. The main result from measuring the water content was that the mean water content of tendons was 61.8% (1.6) and of the meniscus 61.2% (1.3) and both were significantly lower than in other tissues (p < 0:05).

4. Discussion Navicular disease has been diagnosed even without radiographic evidence of lesions in the navicular fibrocartilage (Ackerman et al., 1977). Eight horses in this

study were diagnosed clinically as having pain in the navicular area, but there were no gross pathological lesions on post-mortem examination. In addition, seven horses had lesions of the navicular fibrocartilage, with no history of lameness or other signs of pain in the area. Slight yellowish discoloration of the distal and proximal ridges of the palmar surface of the navicular bone is thought to be a normal ageing change (Colles, 1979). However, gross pathological changes, including ulceration of the fibrocartilage and adhesions between the DDFT and the navicular fibrocartilage, are usually associated with clinical signs of navicular disease (Wright et al., 1998). We have identified for the first time biochemical differences in the extracellular matrix composition of navicular hyaline, fibrocartilage and tendon between healthy horses, animals which are lame but without lesions, animals which are sound with lesions and lame animals with lesions of the fibrocartilage. It is possible that sound horses with lesions might have developed navicular-type lameness if they had lived longer, and equally horses with pain in the navicular area might have developed lesions later on in their life. In this study, erosion of navicular fibrocartilage was reflected in biochemical changes in the extracellular matrix of hyaline cartilage. The COMP content of hyaline cartilage and tendon in horses with navicular disease was significantly higher than in that of sound horses. Interestingly, this response was greater in hyaline cartilage than in fibrocartilage. The increase in COMP production may be an attempt to repair the tissue. COMP levels were increased in fibrocartilage of lame horses as well when compared with fibrocartilage of sound horses, but this difference was not statistically significant. It has been hypothesised that increased levels of COMP are produced in response to loading (Smith et al., 1997). To date, there are no reports in the literature of the navicular hyaline cartilage showing any changes in navicular disease, and direct communication between the navicular bursa and the DIP-joint is rare (Bowker et al., 1993). However, in navicular disease, changes observed in fluid from the navicular bursa are also seen in the synovial fluid of the DIP-joint (Viitanen et al., 2001). One possible explanation for the biochemical changes observed in the hyaline cartilage is that any abnormal pressure exerted on the bone, from a palmar direction, will be transmitted to the dorsal surface, and the hyaline cartilage could therefore be subjected to the same forces leading to the similar biochemical changes as those seen in navicular fibrocartilage. It is also possible that some smaller molecules, such as the pro-inflammatory cytokines arising from erosion sites, may pass through the navicular membrane and thus cause changes in the DIP-joint as well. Both hyaline cartilage and fibrocartilage of sound and lame horses with fibrocartilage lesions had a significantly lower GAG content than comparable cartilage

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

collected from sound and lame horses without lesions. Loss of proteoglycan from cartilage is associated with osteoarthritis. These results indicate that loss of GAG is associated with loss of extracellular matrix, and not pain or lameness, which are the main clinical reasons for diagnosing navicular disease. Interestingly, GAG content was significantly lower in navicular hyaline cartilage in horses with lesions in their navicular fibrocartilage and DDFT, even though no gross pathological lesions were found in the hyaline cartilage itself. This indicates that the disease, which affects the fibrocartilage, may also induce biochemical changes in associated tissues. Analysis of the ÔaggrecanaseÕ and metalloproteinasegenerated cleavage fragments of aggrecan, the main proteoglycan in cartilaginous tissues, did not indicate any variations associated with disease. The most abundant proteoglycan in both hyaline and fibrocartilage is aggrecan. This is lost from tissues by cleavage at two principal sites near to the G1 domain by activities of both the ÔaggrecanaseÕ and metalloproteinase families of enzymes. Although this immunological technique is semi-quantitative, it can reveal major changes in the expression of these fragments (Bird et al., 2000). Disease-related changes in the expression of these epitopes in arthritic cartilage have been previously reported (Bonassar et al., 1997). The lack of change in expression with the onset of disease is thought to be because these changes, which occur principally at the lesion site, are too subtle to be detected by this type of analysis. Cartilage destruction in osteoarthritis (OA) is associated with increased levels of several matrix metalloproteinases (MMPs), including the gelatinases MMP-2 and MMP-9. While increases in some MMPs may be destructive, up-regulation of others may result from increases in normal tissue turnover (Thompson et al., 2001). MMP-2 amount was increased in navicular hyaline cartilage, in fibrocartilage and in DDFT of lame horses with lesions even though the difference was not significant in the case of fibrocartilage. The MMP-2 amount was significantly higher in all tendon tissues when compared to the other tissue types. This could indicate higher turnover in tendon tissues due to higher forces in the deep digital flexor tendon in the navicular area. High relative activity of MMP-9 is observed in osteoarthritis and rheumatoid arthritis. As much as 79% of tendons of lame horses had MMP-9 present and the relative amounts were higher than in tendons of sound horses. This may possibly explain some of the destructive processes in tissues. Increased pressure of the tendon on the bone may have a role in pathogenesis of navicular disease (Pool et al., 1989). Some clinical cases, which have DDFT lesions in the absence of radiographically discernible bone pathology have been described (Wright et al., 1998). Cartilage analysed in this study was obtained from the entire palmar and dorsal surface of the navicular

119

bone. Regional variation in the biochemistry of equine articular cartilage is well known (Brama et al., 2000). Most lesions in navicular disease occur near the sagittal ridge of the navicular bone (Turner, 1986). It is possible that if the samples had been collected only from the affected areas, the differences between the groups would have been more obvious. However, this amount of tissue is insufficient to complete all the analyses in this study. In this study, the cellularity of fibrocartilage decreased with age suggesting that this is a common response in cartilagenous tissues (Platt et al., 1998). A decrease in GAG content with age was also observed in both fibrocartilage and hyaline cartilage. However, Platt et al. (1998) found no change in GAG content of hyaline cartilage relative to tissue dry weight. The reasons for the difference between these findings are unclear, but may be attributable to the number of adolescent animals used in the studies and the statistical analyses used. Age also has an influence on MMP-9 expression in these tissues. This enzyme was identified in all tissues in 40% of adult horses, but was not found in any tissues from adolescent horses. The reasons for this increase with age are unclear, but are consistent with a proposed role in tissue maintenance (Thompson et al., 2001). The biochemistry of navicular fibrocartilage was compared to other fibrocartilages since these are thought to vary depending on anatomical locations and may consequently respond differently to disease processes. The COMP content of the meniscus was higher than in collateral cartilage and navicular fibrocartilage suggesting that meniscus is adapted to resist higher loads than the other two fibrocartilages. GAG levels were lowest in collateral cartilage, which was the only non-compressional fibrocartilage analysed. Although MMP-9 was identified in 40% of adult healthy fibrocartilage, none was found in collateral cartilage or meniscus. In this study we have identified subgroups within animals present with navicular disease. We have also shown that biochemical changes occur in tissues associated with fibrocartilage from diseased joints. Horses with navicular disease have increased amounts of COMP in their navicular hyaline cartilage. MMP-2 amounts are increased in the DDFT and navicular hyaline cartilage, and GAG content decreased in navicular hyaline and fibrocartilage in diseased joints. GAG content also decreased in sound horses, which had lesions in their navicular fibrocartilage. This study has associated biochemical changes in the articular cartilage of the DIP-joint with changes in the palmar aspect of the navicular bone and the bursa in navicular disease. In this disease there are matrix changes in navicular hyaline and fibrocartilage and the DDFT with potential implications for the clinical significance and management of the condition.

120

M. Viitanen et al. / Research in Veterinary Science 75 (2003) 113–120

Acknowledgements We are grateful to Finnish Foundation of Veterinary Sciences, Research and Science Foundation of Farmos and Home of Rest for Horses for financial support. References Adams, O.R., 1969. In: AdamsÕ Lameness in Horses, third ed. Lea and Febiger, Philadelphia, pp. 119–161. Ackerman, N., Johnson, J.H., Dorn, C.R., 1977. Navicular disease in the horse: risk factors, radiographic changes and response to the therapy. J. Am. Vet. Med. Ass. 154, 410–412. Asquith, R.L., Kivipelto, J., 1994. The Navicular syndrome. J. Equine Vet. Sci. 14, 408–410. Bird, J.L., May, S.A., Bayliss, M.T., 2000. Nitric oxide inhibits aggrecan degradation in explant cultures of equine articular cartilage. Equine Vet. J. 32, 133–139. Bonassar, L.J., Sandy, J.D., Lark, M.W., Plaas, A.H., Frank, E.H., Grodzinsky, A.J., 1997. Inhibition of cartilage degradation and changes in physical properties induced by IL-1 and retinoic acid using matrix metalloproteinases inhibitors. Arch. Biochem. Biophys. 344, 404–412. Bowker, R.M., Rockershouser, S.J., Vex, K.B., Sonea, I.M., Caron, J.P., Kotyk, R., 1993. Immunocytochemical and dye distribution studies of nerves potentially desensitized by injections into the distal interphalangeal joint or the navicular bursa of horses. J. Am. Vet. Med. Assoc. 203, 1708–1714. Brama, P.A., Tekoppele, J.M., Bank, R.A., Barneveld, A., van Weeren, P.R., 2000. Functional adaptation of equine articular cartilage the formation of regional biochemical characteristics up to age one year. Equine Vet. J. 32, 217–221. Colles, C.M., 1982. Navicular disease and its treatment. In Pract. 4, 29–36. Colles, C.M., 1979. Ischaemic necrosis of the navicular bone and its treatment. Vet. Rec. 17, 133–137. Clegg, P.D., Coughlan, A.R., Riggs, C.M., Carter, S.D., 1997. Matrix metalloproteinases 2 and 9 in equine synovial fluids. Equine Vet. J. 29, 343–348. Clegg, P.D., Carter, S.D., 1999. Matrix metalloproteinase-2 and -9 are activated in joint diseases. Equine Vet. J. 31, 324–330. Dyson, S.J., 1986. Problems associated with the interpretation of the results of regional and intra-articular anaesthesia in the horse. Vet. Rec. 12 118, 419–422. Dyson, S.J., Kidd, L., 1993. A comparison of responses to analgesia of the distal interphalangeal joint in 59 horses. Equine Vet. J. 25, 93– 98. Farndale, R.W., Buttle, D.J., Barret, A.J., 1986. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem. Biophys. Acta 883, 173–177. Keegan, K.G., Wilson, D.A., Kreeger, J.M., Ellersieck, M.R., Kuo, M.C., Zhaolin, L., 1996. Local distribution of mepivacaine after distal interphalangeal joint injection in horses. Am. J. Vet. Res. 57, 422–426. Kim, Y.J., Sah, R.L., Doong, J.Y., Grodzinsky, A.J., 1988. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal. Biochem. 74, 168–176. Lark, M.W., Bayne, E.K., Flanagan, J., Harper, C.F., Hoerrner, L.A., Hutchinson, N.I., Singer, I.I., Donatelli, S.A., Weidner, J.R., Williams, H.R., Mumford, R.A., Lohmander, L.S., 1997. Aggrecan degradation in human articular cartilage. Evidence on both

matrix metalloproteinase activity in normal, osteoarthritic and rheumatoid joints. J. Clin. Invest. 100, 93–106. MacGregor, C.M., 1984. Studies on the pathology and treatment of equine navicular disease, PhD thesis, University of Edinburgh. Misumi, K., Vilim, V., Clegg, P.D., Thompson, C.C., Carter, S.D., 2001. Measurement of cartilage oligomeric matrix protein (COMP) in normal and diseased equine synovial fluids. Osteoarthritis Cartilage 9, 119–127. Murray, R.C., Smith, R.K., Henson, F.M., Goodship, A., 2001. The distribution of cartilage oligomeric matrix protein (COMP) in equine carpal articular cartilage and its variation with exercise and cartilage deterioration. Vet. J. 162, 121–128. Platt, D., Bayliss, M.T., 1994. An investigation of the proteoglycan metabolism of mature equine articular cartilage and its regulation by interleukin-1. Equine Vet. J. 26, 297–303. Platt, D., Bird, J.L., Bayliss, M.T., 1998. Ageing of equine articular cartilage: structure and composition of aggrecan and decorin. Equine Vet. J. 30, 43–52. Pool, R.R., Meagher, D.M., Stover, S.M., 1989. Pathophysiology of navicular syndrome. Vet. Clinic North Am. Equine Pract. 5, 109– 129. Raulo, S.M., Maisi, P.S., 1998. Gelatinolytic activity in tracheal epithelial lining fluid and in blood from horses with chronic obstructive pulmonary disease. Am. J. Vet. Res. 59, 818–823. Schumacher, J., Steiger, R., Schumacher, J., deGraves, F., Schramme, M.C., Smith, R., Coker, M., 2000. Effects of analgesia of the distal interphalangeal joint or palmar digital nerves on lameness caused by solar pain in horses. Vet. Surg. 29, 54–58. Schumacher, J., Schumacher, J., de Graves, F., Steiger, R., Schramme, M., Smith, R., Coker, M., 2001. A comparison of the effects of two volumes of local analgesic solution in the distal interphalangeal joint of horses with lameness caused by solar toe or solar heel pain. Equine Vet. J. 33, 265–268. Sepper, R., Konttinen, Y.T., Sorsa, 1994. Gelatinolytic and type IV collagenolytic activity in bronchiectasis. Chest 106, 1129–1133. Smith, R., Zunino, L., Webbon, P., Heinegard, D., 1997. The distribution of cartilage oligomeric matrix protein (COMP) in tendon and its variation with tendon site, age and load. Matrix Biol. 16, 255–271. Stashak, T.S., 1987. Diagnosis of lameness. In: AdamsÕ Lameness in Horses. Lea and Febiger, Philadelphia, p. 146. Thompson, C.C., Clegg, P.D., Carter, S.D., 2001. Differential regulation of gelatinases by transforming growth factor beta-1 in normal equine chondrocytes. Osteoarthritis Cartilage 9, 325– 331. Turner, T.A., 1986. Shoeing principles for the management of navicular disease in horses. J. Am. Vet. Med. Assoc. 189, 298–301. Viitanen, M.V., Bird, J., Maisi, P.S., Smith, R., Tulamo, R.-M., May, S.A., 2000. Differences in the concentration of various synovial fluid constituents between the distal interphalangeal joint, the metacarpophalangeal joint and the navicular bursa in normal horses. Res. Vet. Sci. 69, 63–67. Viitanen, M.V., Bird, J., Makela, O., Schramme, M.C., Smith, R., Tulamo, R.-M., May, S.A., 2001. Synovial fluid studies in navicular disease. Res. Vet. Sci. 71, 201–206. Woessner Jr., J., 1998. The matrix metalloproteinase family. In: Parks, W.C., Mecham, R.P. (Eds.), Matrix Metalloproteinases. Academic Press, San Diego, pp. 1–14. Wright, I.M., 1993. A study of 118 cases of navicular disease: radiological features. Equine Vet. J. 25, 493–500. Wright, I.M., Kidd, L., Thorp, B.H., 1998. Gross, histological and histomorphometric features of the navicular bone and related structures in the horse. Equine Vet. J. 30, 220–234.